gM-negative EHV-mutants without heterologous elements

ABSTRACT

The present invention relates to the field of animal health and in particular of Equine Herpes Viruses (EHV) wherein the gene encoding the protein gM is absent, and which is free of heterologous elements. Further aspects of the invention relate to pharmaceutical compositions comprising said viruses, uses thereof, and methods for the prophylaxis and treatment of EHV infections. The invention also relates to pharmaceutical compositions comprising the combination of EHV-1 and EHV-4 viruses wherein the gene encoding the protein gM is absent and which is free of heterologous elements.

SEQUENCE LISTING

This application contains a sequence listing in accordance with 37C.F.R. 1.821-1.825. The sequence listing accompanying this applicationwhich has been submitted electronically in ASCII format is herebyincorporated by reference in its entirety. Said ASCII copy, created onJul. 25, 2014, is names 01-1372-US-5 SL.txt and is 387,567 bytes insize.

BACKGROUND OF THE INVENTION

The present invention relates to the field of animal health and inparticular of Equine Herpes Viruses (EHV) wherein the gene encoding theprotein gM is absent, and which is free of heterologous elements.Further aspects of the invention relate to pharmaceutical compositionscomprising said viruses, uses thereof, and methods for the prophylaxisand treatment of EHV infections. The invention also relates topharmaceutical compositions comprising the combination of EHV-1 andEHV-4 viruses wherein the gene encoding the protein gM is absent andwhich is free of heterologous elements.

Equine herpesvirus 1 (EHV-1), a member of the Alphaherpesvirinae, is themajor cause of virus-induced abortion in equines and causes respiratoryand neurological disease. Equine herpesvirus 4 (EHV-4) can also inducerespiratory symptoms, abortions or neurological disorder. The entire DNAsequence of both species (EHV-1: Strain Ab4p; EHV-4: Strain NS80567) hasbeen determined (Telford, E. A. R. et al., 1992; Telford, E. A. R. etal., 1998). However, only few genes and gene products have beencharacterized in regard to their relevance for the virulence andimmunogenic properties of EHV.

Herpesvirus glycoproteins are crucially involved in the early stages ofinfection, in the release of virions from cells, and in the directcell-to-cell spread of virions by fusion of neighboring cells. To date,11 herpes simplex virus type 1 (HSV-1)-encoded glycoproteins have beenidentified and have been designated gB, gC, gD, gE, gG, gH, gI, gJ, gK,gL, and gM. HSV-1 mutants lacking gC, gE, gG, gI, gJ, and gM are viable,indicating that these genes are dispensable for replication in culturedcells. Comparison of the HSV-1 and equine herpesvirus 1 nucleotidesequences revealed that all of the known HSV-1 glycoproteins areconserved in EHV-1. According to the current nomenclature, theseglycoproteins are designated by the names of their HSV-1 homologs. It isknown that EHV-1 gC, gE and gI are not essential for growth in cellculture, whereas gB and gD are essential for virus growth in culturedcells. The contributions of other EHV-1 glycoproteins to replication incultured cells are not known (Flowers, C. C. et al., 1992).Transcriptional and protein analyses have shown that the glycoproteinsgB, gC, gD, gG, gH, and gK are expressed in EHV-1-infected cells.Glycoprotein gM (encoded by gene UL10 [Baines, J. D. et al., 1991;Baines, J. D. et al.,1993]) is the only reported nonessentialglycoprotein which is conserved in all herpesviral subfamilies and hasbeen described for human and murine cytomegalovirus and theGammaherpesvirinae members EHV-2, herpesvirus saimiri, and Epstein-Barrvirus. Like many herpesvirus glycoproteins, HSV-1 gM is present invirions and membranes of infected cells. HSV-1 mutants solely lacking gMgrew to titers in cell culture systems reduced approximately 10-foldrelative to those of wild-type virus and showed a reduced virulence in amurine model (Baines, J. D. et al., 1991; MacLean, C. A. et al., 1993).The EHV-1 gM homolog (gp21/22a; referred to as EHV-1 gM from now on) wasfirst described by Allen and Yeargan (Allen, G. P. et al, 1987) and wasshown to be a major constituent of the virus envelope. Furtherinvestigations revealed that gene 52, the gene homologous to HSV-1 UL10,encodes the 450-amino-acid EHV-1 gM polypeptide (Pilling, A. et al.,1994; Telford, E. A. R. et al., 1992). EHV-1 gM represents a multiplehydrophobic protein which contains eight predicted transmembrane domainsand has been reported to be present in infected cells and in purifiedvirions as an Mr 45,000 protein (Pilling, A. et al., 1994; Telford, E.A. R. et al., 1992).

For control of EHV-1 infections, two different approaches were followed.First, modified live vaccines (MLVs) have been developed, including thestrain RacH (Mayr, A. et al., 1968; Hübert, P. H. et al., 1996), whichis widely used in Europe and the United States. Second, inactivatedvaccines and subunit vaccines based on recombinant expressed viralglycoproteins such as the glycoproteins (g) B, C, D, and H, whichinduced partial protection against subsequent challenge EHV-1 infectionin a murine model. Subunit vaccines comprising said glycoproteins e.g.gB, gC, gD, and gH only poorly protect against reinfection (Awan et al.,1990, Osterrieder et al., 1995, Tewari et al., 1994, Stokes et al,1996).

The following U.S. patent applications are also incorporated byreference herein: U.S. patent application Ser. No. 09/789,495, filedFeb. 16, 2001, U.S. patent application Ser. No. 10/105,828, filed Mar.25, 2002, and U.S. patent application Ser. No. 09/812,720, filed Mar.20, 2001.

The technical problem underlying this invention was to provide improvedvaccines which protect better against EHV infection than prior artvaccines.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Generation of a gM negative EHV-1 RacH virus without foreignsequences (HΔgM-w)

This figure shows the map of viruses and plasmids used for theconstruction of HΔgM-w. “First-generation” HΔgM virus has previouslybeen constructed by either inserting the Escherichia coli lacZ(HΔgM-lacZ) or the green fluorescent protein (GFP) expression cassette(HΔgM-GFP). The BamHI map of EHV-1 strain RacH is shown (A) and theBamHI-HindIII fragment containing the gM-ORF is magnified showing thegenomic organization of the region (B). The gM-negative virus, HΔgM-GFPcarries a GFP-expression cassette, replacing the major part of the EHV-1gM gene. The GFP-specific probe, that was used in Southern blots, isdepicted (C). Plasmid pBH3.1 carries the EHV-1 BamHI-HindIII fragment ofinterest and was used to construct plasmid pXuBaxA. After cotransfectionof DNA of HΔgM-GFP with plasmid pXuBaxA resulted HΔgM-w (D). Restrictionsites: BamHI-B, HindIII-H, SphI-S, HincII-Hc, ApaI-A, PstI-P

FIG. 2: Southern blot of gM-deleted EHV-1 virus without foreignsequences (HΔgM-w).

DNA of RacH, HΔgM-GFP and of HΔgM-w was cleaved with BamHI, HindIII orPstI and analyzed with a GFP-specific probe (GFP) or the EHV-1BamHI-HindIII fragment of pBH3.1 (pBH3.1). DNA-hybrids were detected bychemoluminescence using CSPD. Molecular weight marker sizes (Biolabs)are given in kbp on the left margin. The arrow points to a barelyvisible specific hybrid.

FIG. 3: Generation of a gM negative EHV-4 virus without foreignsequences (E4ΔgM-w).

In this figure, a BamHI map of EHV-4 strain NS80567 is depicted. Theenlarged BamHI-e fragment encompasses the gM- and neighboring ORFs (A).Plasmid constructs and priming sites are depicted (B). Plasmid pgM4GFP+was used for the generation of E4ΔgM-GFP, the GFP-positive and gMnegative EHV-4 (B, C). Recombination of DNA of E4ΔgM-GFP with eitherplasmid pgM4R (B), containing 3.109 bp of EHV-4 sequences including thegM-ORF, resulted in E4RgM, the gM-repaired EHV-4 (A), or with plasmidpgM4w (B) resulted in E4ΔgM-w, the GFP- and gM-negative EHV-4 (D).Restriction sites: BamHI-B, PstI-P, EcoRI-E, SalI-Sa, MluI-M, AsnI-As,EcoRV-EV

FIG. 4: Southern blot of a gM-deleted EHV-4 virus without foreignsequences (E4ΔgM-w).

DNA of EHV-4, E4RgM, E4ΔgM-w and E4ΔgM-GFP were cleaved with PstI, EcoRVor HindIII as indicated and DNA-fragments blotted onto nylon membranes.Parallel membranes were either hybridized with GFP-specific sequences orwith a probe, named gM3.1, containing the EHV-4 specific sequences takenout of plasmid pgM4R (FIG. 3). DNA hybrids were detected bychemoluminescence using CSPD. Molecular weight marker sizes (Biolabs)are given in kbp.

FIG. 5: Growth characteristics of the gM deleted EHV-4 virus, E4ΔgM-w.

Cells were infected with an MOI of 2 of the different viruses, as listedin the box. Kinetics of virus growth are depicted as virus titersdetermined in supernatants of infected cells (extracellular activity) orwithin infected cells (intracellular activity) relative to the timepoint indicated. Shown are the means of two individual experiments,standard deviations are given as error bars.

FIG. 6A: Plaque sizes of E4ΔgM-w. Vero or Vero-gM cells in 6-well plateswere infected with 50 PFU of either EHV-4, E4RgM, E4ΔgM-GFP or E4ΔgM-w.Maximal diameters of 150 respective plaques were determined and averageplaque sizes are given in %, relative to sizes of wildtype plaques, thatwere set 100%. Standard deviations are given as error bars.

FIG. 6B illustrates the plaques that were stained by indirectimmunofluorescence (anti-gD Mab 20C4, 1:1000) at day 4 p.i. and analyzedin an Axioscope (x100, Zeiss, Germany). Pictures were scanned anddigitally processed.

FIG. 7: EHV-4 virus penetration into Vero cells.

Penetration of EHV-4, E4RgM, E4ΔgM-w and E4ΔgM-GFP produced on noncomplementing Vero cells (A) or on complementing Vero-gM cells (B) intoVero cells. At given time points the penetration efficiency wasdetermined as the percentage of the number of plaques present aftercitrate treatment relative to that of plaques present after controltreatment. Means of two independent experiments are given. Standarddeviations are depicted as error bars.

FIG. 8: PCR primer sequences (SEQ ID NOS 3-10 , respectively, in orderof appearance) and location of amplificates within the EHV-4 genome.

Sequences representing restriction enzyme recognition sites are printedin bold and the respective enzymes are listed. PCR products wereinserted into the given vectors, resulting in plasmids pCgM4, pgM4RpgM4Del1 or pgM4Del2, as indicated. The location of a fragment withinthe EHV-4 genome is given relative to the sequence determined by Telfordet al. (1998) (SEQ ID NO:2).

FIG. 9: Western Blot analysis of horse sera.

Lysates of EHV-1 gM expressing cell line ccgM and Rk13 cells were eitherheated for 2 min at 56° C. (1,2) or for 5 min at 95° C. (3) (gM ishighly hydrophobic and known to aggregate upon boiling, so that it doesnot enter the separating gel anymore). Identical blots were incubatedwith various horse sera (1:3000) or the anti-gM rabbit serum. Arrowspoint to gM specific reactivity in boiled and unboiled samples.Neutralizing test titers (NT) are given for sera, that were obtainedfrom the virological diagnostic unit.

FIG. 10:

(A) Comparison of EHV-1 gM to EHV-4 gM using an anti-EHV-1 gM polyclonalantibody. Cells were infected with the indicated viruses (MOI of 0.5-1)and cell lysates were prepared at the stated time points p.i. EHV-1 or-4 virions were purified by repeated centrifugation through a sucrosecushion (30%) and resuspended in PBS. Samples were mixed with buffercontaining 5% 2-mercaptoethanol and then either heated to 99° C. for 5min or left at 4° C. Proteins were separated by SDS-10% PAGE and blottedonto nitrocellulose filters. Antibody binding was visualized usinganti-rabbit immunoglobulin G (IgG) peroxidase conjugate (Sigma) followedby ECLTM detection (Pharmacia-Amersham).

(B) Virions were purified of Edmin337 cells infected with EHV-4,E4ΔgM-w, E4ΔgM-GFP or E4RgM and subjected to Western blot analysis asdescribed in (A). gB reactivity of virions was then compared to gMreactivity using anti EHV-1 gB monoclonal antibody 3F6 or the anti EHV-1gM polyclonal antibody.

DESCRIPTION OF THE INVENTION Definitions of Terms Used in theDescription

Before the embodiments of the present invention it must be noted that asused herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, reference to “an EHV virus” includes aplurality of such EHV viruses comprising also all subspecies like EHV1,4 and others, reference to the “cell” is a reference to one or morecells and equivalents thereof known to those skilled in the art, and soforth. Unless defined otherwise, all technical and scientific terms usedherein have the same meanings as commonly understood by one of ordinaryskill in the art to which this invention belongs.

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods, devices, and materials are nowdescribed. All publications, patents and patent applications mentionedherein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologies asreported which might be used in connection with the invention. Nothingherein is to be construed as an admission that the invention is notentitled to antedate such disclosure by virtue of prior invention.

The term “EHV” as used herein refers to all equine herpes viruses suchas species EHV-1 and EHV-4 within the family Alphaherpesvirinae. Theterms EH-virus, EH virus EHV virus, EHV-virus and EHV all relate toequine herpes viruses.

Virulence: “virulence” as used herein relates to an EH-virus capable ofpropagating in the target host species (i.e. horses) with the potentialto induce subclinical and clinical disease characterized by respiratorysymptoms, abortions or neurological disorders. Examples of virulent EHVare wild-type viruses inducing strong clinical symptoms. Examples ofvirulence factors are hemolysins (lysing red blood cells) or adhesins(proteins by which the pathogen can adhere to the host and initiatecolonisation or invasion). More specifically, “virulence” here meansthat the gM gene product, a major constituent of the virus envelope,enables the virus to penetrate the host and is involved in cell-to-cellspread.

Attenuation: “An attenuated EH-virus” as used herein is relates toinfectious EHV which do not cause EHV-associated subclinical or clinicaldisease. In particular according to the invention, such attenuatedEH-viruses are EHV which can replicate and do not express gM.

A “functional variant” of the EH-virus according to the invention is EHVvirus which possesses a biological activity (either functional orstructural) that is substantially similar to the EHV according to theinvention. The term “functional variant” also includes “a fragment”, “afunctional variant”, “variant based on the degenerative nucleic acidcode” or “chemical derivative”. Such a “functional variant” e.g. maycarry one or several nucleic acid substitutions, deletions orinsertions. Said substitutions, deletions or insertions may account for10% of the entire sequence. Said functional variant at least partiallyretains its biological activity, e.g. function as an infectious clone ora vaccine strain, or even exhibits improved biological activity.

A “variant based on the degenerative nature of the genetic code” is avariant resulting from the fact that a certain amino acid may be encodedby several different nucleotide triplets. Said variant at leastpartially retains its biological activity, or even exhibits improvedbiological activity.

A “fusion molecule” may be the DNA molecule or infectious EHV virusaccording to the invention fused to e.g. a reporter such as aradiolabel, a chemical molecule such as a fluorescent label or any othermolecule known in the art.

As used herein, a “chemical derivative” according to the invention is aDNA molecule or infectious EHV clone according to the inventionchemically modified or containing additional chemical moieties notnormally part of the molecule. Such moieties may improve the molecule'ssolubility, absorption, biological half-life etc.

A molecule is “substantially similar” to another molecule if bothmolecules have substantially similar nucleotide sequences or biologicalactivity. Thus, provided that two molecules possess a similar activity,they are considered variants as that term is used herein if thenucleotide sequence is not identical, and two molecules which have asimilar nucleotide sequence are considered variants as that term is usedherein even if their biological activity is not identical.

The term “vaccine” as used herein refers to a pharmaceutical compositioncomprising at least one immunologically active component that induces animmunological response in an animal and possibly but not necessarily oneor more additional components that enhance the immunological activity ofsaid active component. A vaccine may additionally comprise furthercomponents typical to pharmaceutical compositions. The immunologicallyactive component of a vaccine may comprise complete virus particles ineither their original form or as attenuated particles in a so calledmodified live vaccine (MLV) or particles inactivated by appropriatemethods in a so called killed vaccine (KV). In another form theimmunologically active component of a vaccine may comprise appropriateelements of said organisms (subunit vaccines) whereby these elements aregenerated either by destroying the whole particle or the growth culturescontaining such particles and optionally subsequent purification stepsyielding the desired structure(s), or by synthetic processes includingan appropriate manipulation by use of a suitable system based on, forexample, bacteria, insects, mammalian or other species plus optionallysubsequent isolation and purification procedures, or by induction ofsaid synthetic processes in the animal needing a vaccine by directincorporation of genetic material using suitable pharmaceuticalcompositions (polynucleotide vaccination). A vaccine may comprise one orsimultaneously more than one of the elements described above.

The term “vaccine” as understood herein is a vaccine for veterinary usecomprising antigenic substances and is administered for the purpose ofinducing a specific and active immunity against a disease provoked byEHV. The EHV vaccine according to the invention confers active immunitythat may be transferred passively via maternal antibodies against theimmunogens it contains and sometimes also against antigenically relatedorganisms.

Additional components to enhance the immune response are constituentscommonly referred to as adjuvants, like e.g. aluminumhydroxide, mineralor other oils or ancillary molecules added to the vaccine or generatedby the body after the respective induction by such additionalcomponents, like but not restricted to interferons, interleukins orgrowth factors.

A “vaccine composition” or “pharmaceutical composition” essentiallyconsists of one or more ingredients capable of modifying physiological eg immunological functions of the organism it is administered to, or oforganisms living in or on the organism. The terms include, but are notrestricted to antibiotics or antiparasitics, as well as otherconstituents commonly used to achieve certain other objectives like, butnot limited to, processing traits, sterility, stability, feasibility toadminister the composition via enteral or parenteral routes such asoral, intranasal, intravenous, intramuscular, subcutaneous, intradermalor other suitable route, tolerance after administration, controlledrelease properties.

Disclosure of the Invention

The invention overcomes the difficulties and prejudice in the art thatan equine herpes virus cannot be generated free of foreign sequences.The solution to the above technical problem is achieved by thedescription and the embodiments characterized in the claims. By usingthe methods according to the invention, EH-viruses of superior qualityfor use in vaccines are provided. The central coding sequence for theprotein gM is eliminated in a way that the remaining gM carboxyterminalsequences are in a different reading frame than the aminoterminalsequences. The neighboring gene for the essential protein UL9 homolog(gene 53), its orientation and overlap with the gene coding for theprotein gM requires that a minimal nucleotide sequence of the gene forgM must remain to allow the expression of gene 53 and thereby retainvirus viability. Therefore, an EHV according to the invention relates toEHVs that are characterized in that the gene coding for the protein gMis deleted in a way that the expression of the gene coding for the UL9homolog (gene 53) is not affected. The term “not affected” does notrelate to certain quantity or qualitative properties of UL9 but simplymeans that the expression of the gene is not affected as long as saidprotein is expressed by the virus and present in an essentiallysufficient amount for the viability of the virus.

The long lasting need in the art for a vaccine comprising recombinantequine herpesvirus 4 is satisfied by the present invention whichovercomes major difficulties in the art. The EHV-1 and EHV-4 virusesaccording to the invention may advantageously be used, for example, in avaccine.

Hence, in a first important embodiment, the invention relates to arecombinant Equine Herpes Virus (EHV) wherein the gene encoding proteingM, and therefore gM itself, is absent, characterized in that it is freeof heterologous elements. “Free of heterologous elements” means that noforeign sequence, i.e. no non-EHV sequence, such as a lacZ- orGFP-encoding cassette, is present in the coding sequence for said virusaccording to the invention (a so-called “white clone”). Thus, the EHVaccording to the invention is entirely encoded by EHV sequences. The EHVaccording to the invention is free of bacterial elements or nucleicacids encoding said bacterial elements. Furthermore, almost the entirecoding sequence for the gM protein and therefore the encodedabove-mentioned gM protein is eliminated. Thus, preferably, said EHVaccording to the invention is characterized in that the protein gM isabsent due to deletion of the gene coding for the protein gM. However,as set out supra, “the gene encoding protein gM is absent” also requiresthat a minimum gM sequence remains so that at least the overlapping gene53 sequence is still present, while the remaining gM sequences may bedeleted (see infra). This may all be accomplished by molecular biologytechniques (see infra) so that recombinant EHV are generated.

The use of lacZ as a marker for successful deletion of the gM gene ofEHV-1 or 4 did not lead to successful generation of viruses according tothe invention (see Examples 1, 2). The inventors therefore developed aninventive method to obtain said virus. An EH-virus was constructed inwhich the gM gene was deleted by insertion of a cassette containing theGFP marker. This approach surprisingly allowed the differentiationbetween input virus (green fluorescent plaques) and new recombinantplaques (non-fluorescent plaques).

Preferred is an EHV obtainable by a method comprising the steps of:

-   -   a) isolating a wild-type EHV;    -   b) establishing a plasmid encoding the EHV gM gene, optionally        with flanking sequences;    -   c) generating a complementing cell line expressing gM or parts        thereof;    -   d) establishing an EH virus carrying a GFP-encoding cassette        insert in its gM coding sequence by co-transfecting the        complementing cell line of step b) with EHV-nucleic acid and a        plasmid encoding gM which is interrupted by a GFP-encoding        cassette insert;    -   e) deleting the GFP-encoding cassette; and    -   f) selecting for the EHV clones wherein the GFP-encoding        cassette is successfully deleted.    -   g) “lacZ” is known to the artisan as the gene encoding        β-galactosidase. According to the invention, “GFP” relates to        green fluorescent protein (GFP) produced by the bioluminescent        jellyfish (Chalfie et al., 1994).

“Complementing cell line” refers to a cell line, into which a genenormally not present in the cell line genome is introduced and expressedconstitutively. Useful cell lines include, but are not limited to rabbitkidney cell line Rk13, cell line cc (Seyboldt et al., 2000) or the Verocell lines (ATCC catalogue #CRL-1586), such as clone 1008, as alsodisclosed in Examples 1 and 2, and any other cell line known to theartisan. Usually it can be selected for cell clones expressing thisadditional protein. This cell line expresses the gene which is deletedin the virus, complementing this deficiency, and enables the growth ofthe virus after gene deletion.

Standard molecular biology methods of use of restriction enzymes,ligation, PCR, transfection etc. are known in the art (see e.g. Sambrooket al. (1989). Molecular Cloning: A Laboratory Manual, 2nd ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.)

Preferably, such EHV according to the invention is characterized in thatit is EHV-1. More preferred, the EHV-1 according to the invention ischaracterized in that 850-1100 bp of the 1332 bp gM open reading frameare deleted. Even more preferred, the EHV-4 according to the inventionis characterized in that 900-1000 bp of the gM open reading frame aredeleted. More preferred also, the EHV-1 according to the invention ischaracterized in that 960-970 bp of the gM open reading frame aredeleted (960, 961, 962, 963, 964, 965, 966, 967, 968, 969 or 970 bp).Most preferred, the EHV-1 according to the invention is characterized inthat 962 bp of the gM open reading frame are deleted.

More preferred, the EHV-1 according to the invention is characterized inthat the coding sequence for gM is deleted except for 150-200 base pairs(bp) of the coding sequence encoding the C-terminal portion of gM and150-250 bp of the coding sequence encoding the N-terminal portion of gM.In this more preferred embodiment, the coding sequence of gM is deleted,only nucleotides 93118-93267 to 93118-93317 of the sequence encoding theC-terminal portion of gM and nucleotides 94223-94472 to 94323-94472 ofthe coding sequence encoding the N-terminal portion of gM remain. Thus,more preferred is an EHV-1 according to the invention characterized inthat nucleotides 93268-93318 to 94222-94322 (encoding the core portionof gM) are deleted (numbering according to Telford, 1992, SEQ ID NO:1).Within the given ranges, any number of nucleotides may be deleted. Thus,according to the invention, the deletions may start no lower thannucleotide position 93268, but has to begin at position 93318. Thedeletion may end as early as position 94222, but no later than position94322. Thus, a preferred EHV-1 according to the invention ischaracterized in that nucleotides 93268 to 94322 of the gM codingsequence as corresponding to SEQ ID NO:1 are deleted. Any combination iswithin the scope of the invention, such as 93272 to 94312, 93300 to94300 and so forth.

Even more preferred, the EHV-1 according to the invention ischaracterized in that the coding sequence for gM is deleted except for160-190 bp of the coding sequence encoding the C-terminal portion of gMand 190-220 bp of the coding sequence encoding the N-terminal portion ofgM. In this more preferred embodiment, the coding sequence of gM isdeleted, only nucleotides 93118-93277 to 93118-93307 of the sequenceencoding the C-terminal portion of gM and nucleotides 94253-94472 to94283-94472 of the coding sequence encoding the N-terminal portion of gMremain. Thus, more preferred is an EHV-1 according to the inventioncharacterized in that nucleotides 93278-93308 to 94252-94282 (encodingthe core portion of gM) are deleted (numbering according to SEQ IDNO:1).

More preferred also, the EHV-1 according to the invention ischaracterized in that the coding sequence for gM is deleted except for180 to 190 (180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190) bp ofthe coding sequence encoding the C-terminal portion of gM codingsequence and 200 to 210 (200, 201, 202, 203, 204, 205, 206, 207, 208,209 or 210) bp of the coding sequence encoding the N-terminal portion ofgM. In this more preferred embodiment, the coding sequence of gM isdeleted, only nucleotides 93118-93297 to 93118-93307 (93297, 93298,93299, 93300, 93301, 93302, 93303, 93304, 93305, 93306, 93307) of thesequence encoding the C-terminal portion of gM and nucleotides94263-94472 to 94273-94472 (94263, 94264, 94265, 94266, 94267, 94268,94269, 94270, 94271, 94272, 94273) of the coding sequence encoding theN-terminal portion of gM remain. Thus, more preferred is an EHV-1according to the invention characterized in that nucleotides 94298-94308to 94262-94272 (encoding the core portion of gM) are deleted (numberingaccording to SEQ ID NO:1). This means, that any nucleotides inside theabove-mentioned remaining nucleotides may be deleted according to theinvention, e.g. nucleotides 94299-94263 or 94299-94264 or 94300-94272 orany combination thereof.

Most preferred, the EHV-1 according to the invention is characterized inthat the coding sequence for gM is deleted except for 184 bp of thecoding sequence encoding the C-terminal portion of gM coding sequenceand 209 bp of the coding sequence encoding the N-terminal portion of gM.In this most preferred embodiment, the coding sequence of gM is deleted,only nucleotides 93118-93301 of the sequence encoding the C-terminalportion of gM and nucleotides 94264-94472 of the coding sequenceencoding the N-terminal portion of gM remain. Thus, most preferred is anEHV-1 according to the invention characterized in that nucleotides 94263to 93302 (encoding the core portion of gM) are deleted (numberingaccording to SEQ ID NO:1). In this most preferred embodiment, 962nucleotides of the sequence encoding gM are deleted. This is exemplifiedin a non-limiting manner in Example 1.

Also more preferred is an EHV-1 characterized in that gM is deleted andit is free of heterologous elements and it is a recombinant variantbased on a strain selected from the group of AB69 (ATCC VR2581), EHV-1Ts-mutant ECACC V99061001, KyA, KyD, Ab1, Ab4, RacH, RacL11 or RacM ofEHV-1 and no heterologous elements such as GFP- or lacZ-elements arepresent. Also more preferred, an EHV-1 according to the invention ischaracterized in that gM is deleted and it is free of heterologouselements such as GFP- or lacZ-elements and it is a recombinant variantbased on strain RacH of EHV-1. Most preferred, an EHV-1 according to theinvention is characterized in that gM is deleted and it is free ofheterologous elements such as GFP- or lacZ-elements and it is theRacH-based recombinant variant isolate HΔgM-w as disclosed in Example 1.Said EHV-1 HΔgM-w according to the invention was deposited at the“Centre for Applied Microbiology and Research (CAMR) and EuropeanCollection of Cell Cultures (ECACC)”, Salisbury, Wiltshire SP4 0JG, UK,as patent deposit according to the Budapest Treaty. The date of depositwas Oct. 16, 2002, the preliminary identification reference isH-delta-gM-w, and the accession number given by the internationaldepository authority ECACC/CAMR is 02101663. Also preferred are EHV-1having all of the identifying characteristics of said deposited EHV-1.

All before-mentioned EHV-1 have superior properties over viruses withheterologous elements such as GFP. Said EHV-1 according to the inventionhave an advantageously higher extracellular infectivity than those stillcomprising heterologous elements. This is exemplified in FIG. 5 (e.g.between 4 and 12 hours).

Until the present invention was made, no one in the art was able togenerate a recombinant EHV-4 virus which may be used as a vaccine. EHV-1and EHV-4 are homologous and cross-reactive to some degree. However,there was a long need in the art for attenuated EHV-4 viruses as EHV-1does not appear to provide sufficient protection against EHV-4infection. Thus, preferably, an EHV according to the invention ischaracterized in that it is EHV-4. More preferred, the EHV-4 accordingto the invention is characterized in that 900-1150 bp of the 1332 bp gMopen reading frame are deleted. Even more preferred, the EHV-4 accordingto the invention is characterized in that 1000-1150 bp of the gM openreading frame are deleted. More preferred also, the EHV-1 according tothe invention is characterized in that 1110-1115 bp of the gM openreading frame are deleted (1110, 1111, 1112, 1113, 1114 or 1115 bp).Most preferred, the EHV-1 according to the invention is characterized inthat 1110 bp of the gM open reading frame are deleted.

More preferred, the EHV-4 according to the invention is characterized inthat the coding sequence for gM is deleted except for 0-50 base pairs(bp) of the coding sequence encoding the C-terminal portion of gM and150-250 bp of the coding sequence encoding the N-terminal portion of gM.In this more preferred embodiment, the coding sequence of gM is deleted,only nucleotides 92681-92680 to 92681-92730 of the sequence encoding theC-terminal portion of gM and nucleotides 93766-94033 to 93866-94033 ofthe coding sequence encoding the N-terminal portion of gM remain. Thus,more preferred is an EHV-4 according to the invention characterized inthat nucleotides 92681-92731 to 93765-93865 (encoding the core portionof gM) are deleted (numbering according to SEQ ID NO:2). Within thegiven ranges, any number of nucleotides may be deleted. Thus, accordingto the invention, the deletions may start no lower than nucleotideposition 92681, but has to begin at position 92731. The deletion may endas early as position 93765, but no later than position 93865. Thus,preferably, an EHV-4 according to the invention is characterized in thatnucleotides 92681 to 93865 of the gM coding sequence as corresponding toTelford positions (1998) (SEQ ID NO:2) are deleted. Any combination iswithin the scope of the invention, such as 92672 to 93801, 92700 to93800 and so forth.

Even more preferred, the EHV-4 according to the invention ischaracterized in that the coding sequence for gM is deleted except for10-40 bp of the coding sequence encoding the C-terminal portion of gMand 190-220 bp of the coding sequence encoding the N-terminal portion ofgM. In this more preferred embodiment, the coding sequence of gM isdeleted, only nucleotides 92681-92690 to 92681-92720 of the sequenceencoding the C-terminal portion of gM and nucleotides 93806-94033 to93836-94033 of the coding sequence encoding the N-terminal portion of gMremain. Thus, more preferred is an EHV-4 according to the inventioncharacterized in that nucleotides 92691-92721 to 93805-93835 (encodingthe core portion of gM) are deleted (numbering according to SEQ IDNO:2).

More preferred also, the EHV-4 according to the invention ischaracterized in that the coding sequence for gM is deleted except for30 to 40 (30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40) bp of the codingsequence encoding the C-terminal portion of gM coding sequence and 200to 210 (200, 201, 202, 203, 204, 205, 206, 207, 208, 209 or 210) bp ofthe coding sequence encoding the N-terminal portion of gM. In this morepreferred embodiment, the coding sequence of gM is deleted, onlynucleotides 92681-92710 to 92681-92720 (92710, 92711, 92712, 92713,92714, 92715, 92716, 92717, 92718, 92719, 92720) of the sequenceencoding the C-terminal portion of gM and nucleotides 93816-94033 to93826-94033 (93824, 93825, 93826, 93827, 93828, 93829, 93830, 93831,93832, 93833, 93834) of the coding sequence encoding the N-terminalportion of gM remain. Thus, more preferred is an EHV-4 according to theinvention characterized in that nucleotides 92711-92721 to 93823-93833(encoding the core portion of gM) are deleted (numbering according toSEQ ID NO:2). This means, that any nucleotides inside theabove-mentioned remaining nucleotides may be deleted according to theinvention, e.g. nucleotides 94299-94257 or 94299-94256 or 94300-94257 orany combination thereof.

Most preferred, the EHV-4 according to the invention is characterized inthat the coding sequence for gM is deleted except for 34 bp of thecoding sequence encoding the C-terminal portion of gM coding sequenceand 209 bp of the coding sequence encoding the N-terminal portion of gM.In this most preferred embodiment, the coding sequence of gM is deleted,only nucleotides 92681-92714 of the sequence encoding the C-terminalportion of gM and nucleotides 93825-94033 of the coding sequenceencoding the N-terminal portion of gM remain. Thus, most preferred is anEHV-4 according to the invention characterized in that nucleotides 92715to 93824 (encoding the core portion of gM) are deleted (numberingaccording to SEQ ID NO:2). In this most preferred embodiment, 1110nucleotides of the sequence encoding gM are deleted. This is exemplifiedin a non-limiting manner in Example 2.

Also more preferred, an EHV-4 according to the invention ischaracterized in that gM is deleted and it is free of heterologouselements such as GFP- or lacZ-elements and it is a recombinant variantbased on strain MSV Lot 071398 of EHV-4. Most preferred, an EHV-4according to the invention is characterized in that gM is deleted and itis free of heterologous elements such as GFP- or lacZ-elements and it isbased on strain MSV Lot 071398 and isolate E4ΔgM-4 as disclosed inExample 2. Said EHV-1 HΔgM-w according to the invention was deposited atthe “Centre for Applied Microbiology and Research (CAMR) and EuropeanCollection of Cell Cultures (ECACC)”, Salisbury, Wiltshire SP4 0JG, UK,as patent deposit according to the Budapest Treaty. The date of depositwas Jan. 14, 2003, the preliminary identification reference is EHV-4,and the accession number given by the international depositary authorityECACC/CAMR is 03011401. Also preferred are EHV-4 having all of theidentifying characteristics of said deposited EHV-4.

All before-mentioned EHV-4 have superior properties over viruses knownin the prior art as there are no recombinant EHV-4 available in the art.Furthermore, said EHV-4 according to the invention have anadvantageously higher extracellular infectivity than those stillcomprising heterologous elements such as GFP. This is exemplified inFIG. 10 (e.g. at 24 hours).

Another important element of the invention is a nucleic acid coding foran EHV as disclosed supra. The artisan can easily determine thecorresponding sequence by standard molecular biology methods known inthe art.

There was a particular difficulty in the art to obtain the EHV accordingto the invention. The present inventors constructed gM negative EHVviruses by introducing a marker gene (lacZ) into the gM gene. When itwas attempted to remove this cassette, in both EHV-1 and EHV-4 mutantsproduced by lacZ insertion, all clones phenotypically lacZ negativestill contained the lacZ cassette. The inventors therefore developed aninventive method to obtain said viruses. An EH virus was constructed inwhich the gM gene was deleted by insertion of a cassette containing theGFP marker. This approach surprisingly allowed the differentiationbetween input virus (green fluorescent plaques) and new recombinantplaques (non-fluorescent plaques). Also, a Vero cell line (based on Verocell clone 1008) constitutively expressing EHV4-gM was generated by thepresent inventors to overcome the difficulties in the art. Said cellline was generated by transfection of the appropriate gM gene andsubsequent selection for gM-expressing Vero cells. Only said cellsenabled the inventors to replicate EHV4 gM negative virus. SaidgM-complementing Vero cell line according to the invention was depositedat the “Centre for Applied Microbiology and Research (CAMR) and EuropeanCollection of Cell Cultures (ECACC)”, Salisbury, Wiltshire SP4 0JG, UK,as patent deposit according to the Budapest Treaty. The date of depositwas Jan. 28, 2003, the preliminary identification reference is VERO GM,and the accession number given by the international depositary authorityECACC/CAMR is 03012801. Also preferred are cell lines having all of theidentifying characteristics of said deposited VERO GM cell line.

Preferred is a method for obtaining a recombinant EHV, comprising thesteps of:

-   -   a) isolating a wild-type EHV;    -   b) establishing a plasmid encoding the EHV gM gene, optionally        with flanking sequences;    -   c) generating a complementing cell line expressing gM or parts        thereof;    -   d) establishing an EH virus carrying a GFP-encoding cassette        insert in its gM coding sequence by co-transfecting the        complementing cell line of step b) with EHV-nucleic acid and a        plasmid encoding gM which is interrupted by a GFP-encoding        cassette insert;    -   e) deleting the GFP-encoding cassette; and    -   f) selecting for the EHV clones wherein the GFP-encoding        cassette is successfully deleted.

Said above-captioned cells are an important embodiment of the presentinvention. Thus, the invention relates to a cell line for use in amethod according to the invention, characterized in that the geneencoding the protein gM is transfected into said cell line and said cellline expresses gM. The invention preferably relates to a cell lineaccording to the invention, characterized in that it is a cell lineselected from the group of Vero cells (Vero-gM cells), RK-13, and cc(cc-gM).

As disclosed supra for EHV-1, the use of lacZ as a marker instead of GFPin EHV-4 also did not lead to successful generation of viruses accordingto the invention (see in a non-limiting manner in Example 2).“LacZ-positive” cells generally stained less intense on Vero cells thanon Rk13 cells and were thus harder to identify, and the EHV-4 systemreplicated slower than EHV-1 and thus gave less time between plaqueidentification and isolation of viable virus progeny. Therefore, the useof GFP represented the only way to obtain said EHV-4 virus. Theprocedure was carried out as described supra for EHV-1 and surprisinglyalso led to the successful identification of EHV-4 gM deleted virus byvirtue of identifying fluorescent plaques.

The isolation of wild-type EHV is accomplished by collecting lung tissueat necropsy from animals suspected to have been diseased by EHV, andisolating EHV on tissue cells as known in the art. The EHV 1 completegenome sequence has been published by Telford et al. (1992) (SEQ IDNO;1). Likewise, the complete genome sequence for EHV-4 has beenpublished by Telford et al. (1998) (SEQ ID NO:2). The PCR amplificationof DNA sequences by use of specific primers binding to complementarystrands of target DNA flanking the DNA stretch of interest represents astandard molecular biology method. Methods for ligating appropriate DNAsequences into plasmids suitable for the constructions intended, for DNAtransfection into eukaryotic cells, for Southern Blot and Western Blotanalyses, for site-directed excision of DNA fragments via restrictionenzymes and for selection of cell lines expressing the desiredheterologous gene or plasmids harboring the desired gene or virus inwhich a certain gene is deleted are known to the skilled person.Standard molecular biology methods such as above mentioned techniquesare known to the skilled person and can also be found e.g. in Sambrooket al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Bertram, S.and Gassen, H. G. Gentechnische Methoden, G. Fischer Verlag, Stuttgart,New York, 1991).

“Deletion” means the removal of one or several nucleotides or aminoacids.

Another important embodiment of the invention is a pharmaceuticalcomposition or vaccine comprising an EHV according to the invention,optionally in combination with a pharmaceutically acceptable carrier orexcipient.

Also an important part of the present invention is a pharmaceuticalcomposition comprising a nucleic acid according to the invention asdisclosed supra.

Preferably, a vaccine according to the invention refers to a vaccine asdefined above. The term “live vaccine” refers to a vaccine comprising aparticle capable of replication, in particular, a replication activeviral component.

Preferably, a vaccine according to the invention comprises a gM-deletedEHV-1 according to the invention as disclosed supra combined with agM-deleted EHV-4 according to the invention as disclosed supra oroptionally any other antigenetic group and optionally a pharmaceuticallyacceptable carrier or excipient. Said vaccine may be administered as acombined vaccine at the same point in time. Most preferably, saidattenuated EHV-1 according to the invention may be administered firstfollowed by administration of an attenuated EHV-4 according to theinvention three to four weeks later. Most preferably also, saidattenuated EHV-1 according to the invention may be administered incombination with an attenuated EHV-4 according to the invention in atypical vaccination scheme where two or three basic vaccinations aregiven. A typical vaccination scheme of such a vaccine is twovaccinations four weeks apart (basic vaccination), followed by regularboosts every six months. However, any of said vaccines according to theinvention as disclosed supra may also be administered at differentintervals, e.g. every three months.

The artisan may choose to divide the administration into two or moreapplications, which may be applied shortly after each other, or at someother predetermined interval range. Preferably, such interval may be: 1°immunization, 2° immunization approx. 4 weeks thereafter and optionally3° immunization 5-6 months thereafter. Depending on the desired durationand effectiveness of the treatment, vaccines may be administered once orseveral times, also intermittently. The vaccines according to theinvention may be administered to a mare prior to breeding and againduring its pregnancy to prevent EHV-associated abortions. Other horsescan be vaccinated, e.g. once a year. Foals may be vaccinated shortlyafter birth.

The vaccines of the present invention may be applied by different routesof application known to the expert, notably intravenous injection ordirect injection into target tissues. For systemic application, theintravenous, intravascular, intramuscular, intraarterial,intraperitoneal, oral, or intramucosal (e.g. nasal or respiratory sprayor injection) routes are preferred. A more local application can beeffected subcutaneously, intracutaneously, intrapulmonarily or directlyin or near the tissue to be treated (connective-, bone-, muscle-,nerve-, epithelial tissue). A vaccine composition according to theinvention can also be administered via an implant or orally. Mostpreferred is the intramuscular administration.

For preparing suitable vaccine preparations for the applicationsdescribed above, the expert may use known injectable, physiologicallyacceptable sterile solutions. For preparing a ready-to-use solution forparenteral injection or infusion, aqueous isotonic solutions, such ase.g. saline or corresponding plasma protein solutions are readilyavailable. The vaccine preparations may be present as lyophylisates ordry preparations, which can be reconstituted with a known injectablesolution directly before use under sterile conditions, e.g. as a kit ofparts. The final preparation of the vaccine preparations of the presentinvention are prepared for injection, infusion or perfusion by mixingpurified virus according to the invention with a sterile physiologicallyacceptable solution, that may be supplemented with known carriersubstances or/and excipient.

The applied dose of each EH-virus according to the invention present inthe vaccine formulation preferably may be between 104 and 108 TCID50/peranimal, between 105 and 107 TCID50/per animal, most preferably 106TCID50/per animal.

The invention further relates to the use of EHV according to theinvention in the manufacture of a medicament for the prophylaxis and/ortreatment of EHV-associated conditions.

The invention further relates to the use of a nucleic acid according tothe invention in the manufacture of a medicament for the prophylaxisand/or treatment of EHV-associated conditions.

The invention further relates to a method for the prophylaxis and/ortreatment of an animal characterized in that a pharmaceuticalcomposition according to the invention is applied to said animal and thetherapeutic success is monitored.

The invention preferably relates to a method of treating an EHV-infectedequine animal with a gM-deleted EHV according to the invention asdescribed supra, wherein the said attenuated EHV or the vaccinecomposition as disclosed supra is administered to the equine animal inneed thereof at a suitable dose as known to the skilled person and thereduction of EHV symptoms such as viremia and leukopenia and/or coughingand/or pyrexia and/or nasal discharge and/or diarrhea and/or depressionand/or abortion is monitored. Said treatment preferably may be repeated.Thus, the invention relates to a method for the prophylaxis and/ortreatment of an animal characterized in that a pharmaceuticalcomposition according to the invention is applied to said animal and thetherapeutic success is monitored. The treatment may be carried out asdisclosed for the vaccine composition supra.

The invention preferably relates to a method of detecting antibodiesagainst specific structures of infecting EHV-1 or EHV-4 and to a methodof differentiating wild-type infections from the presence of gM deletedEHV-1 or EHV-4 as described above by an immunological methodImmunological methods are known to the expert in the field and include,but are not limited to ELISAs (enzyme-linked immuno-sorbent assay) orSandwich-ELISAs, dot-blots, immunoblots, radioimmunoassays(Radioimmunoassay RIA), diffusion-based Ouchterlony tests, rocketimmunofluorescent assays or Western-blots. Examples for immunologicalmethods are e.g. described in: An Introduction to Radioimmunoassay andRelated Techniques, Elsevier Science Publishers, Amsterdam. TheNetherlands (1986); Bullock et al., Techniques in Immunocytochemistry,Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3(1985); Tijssen, Practice and Theory of Enzyme Immunoassays: LaboratoryTechniques in Biochemistry and Molecular Biology, Elsevier SciencePublishers, Amsterdam, The Netherlands (1985).

Said ELISA may use, but not be confined to the use of immobilized gMgene product or a part of gM gene product or any other EH virus 1 or EHvirus 4 gene product on a plastic surface suitable for ELISA analysis.

An ELISA according to the present invention comprises, but is notlimited to the steps of

-   -   a) immobilizing a gM gene product or a fragment thereof onto a        plastic support    -   b) rinsing the plastic surface with an appropriate washing        buffer (e.g., PBS-Tween)    -   c) adding the samples to selected wells and incubating the ELISA        plate according to standardized methods    -   d) washing the wells of the ELISA plate and adding a suitable        antibody coupled to an enzyme such as HRP (horse radish        peroxidase)    -   e) detecting bound antibody/HRP conjugate by adding a suitable        substrate, followed by photometric read-out of optical density        of individual wells. Suitable antibodies, e.g. rabbit anti horse        Ig, are known in the field.

EXAMPLES

The following examples serve to further illustrate the presentinvention; but the same should not be construed as limiting the scope ofthe invention disclosed herein.

Example 1 gM Deleted EHV-1 Isolates

The gM negative EHV-1 were constructed by either inserting theEscherichia coli lacZ (HΔgM-lacZ) or the green fluorescent protein (GFP)expression cassette (HΔgM-GFP) thereby replacing 74.5% of gM-genesequences. The expression of a marker protein facilitates theidentification and subsequent purification of a recombinant virus. Toavoid the presence of any “non-EHV-1” sequences within the vaccinevirus, it was decided to remove the marker gene sequences and constructanother, second generation gM-negative EHV-1, a “white” HΔgM (HΔgM-w).

To this end, plasmid pXuBaxA was constructed (FIG. 1). At firstrecombination of pXuBaxA-sequences into the lacZ-marked virus HΔgM-lacZwas attempted. In a first step, DNA transfections mediated by thecalcium phosphate method were optimized, such, that several whiteplaques resulted after plating of 100-1000 PFU of transfectionsupernatants. Consequently, several plaques were chosen for purificationof progeny virus and subjected to four to five rounds of isolation ofsingle plaques.

Multiple, independently isolated, phenotypically lacZ-negative viruspopulations were genotypically analyzed by Southern blotting and turnedout to still carry sequences of the lacZ-cassette. Difficulties inisolating truly lacZ-negative virus populations due to “lacZ-silencing”had been anticipated and therefore a great number of phenotypicallylacZ-negative virus populations were purified and analyzed withoutsuccess. Therefore, the strategy of generating a “white” gM-negativeRacH virus was changed by switching to cotransfections with the gMnegative EHV-1, that had been constructed by insertion of a GFPcassette. Using the GFP-expressing virus facilitated the distinctionbetween input virus (green fluorescent plaques) and new recombinantviruses (non fluorescent plaques) and thus increased the efficiency ofisolating phenotypically GFP-negative plaques. Changing the “input”gM-negative RacH was not supposed to influence the genotype of theexpected recombinant virus as (i) both the first generation HΔgM virusesare, apart from the marker, genetically identical and as (ii) the finalgenotype in the region of interest is determined by the recombinationplasmid (pXuBaxA).

For construction of plasmid pXuBaxA (construct necessary to obtain the“white” gM negative EHV-1) the 962 bp ApaI-HincII fragment within the1352 bp open reading frame of EHV-1 gM was removed of plasmid pBH3.1(FIG. 1, section D). Plasmid pBH3.1 carries the EHV-1 BamHI-HindIIIfragment surrounding the gM gene (Seyboldt et al., 2000). To preventexpression of any truncated gM-product, restriction endonucleases ApaIand HincII have been chosen such that after blunt end adjusting andligation the remaining C-terminal gM sequences (183 bp) were not inframe with the remaining N-terminal sequences (208 bp).

EHV-1 gM expressing cell line ccgM (Seyboldt at al., 2000; obtained fromDr. N. Osterrieder) was maintained in minimal essential mediumsupplemented with 5-10% fetal calf serum (Biochrom, γ-irradiated).Homologous recombination into EHV-1 was achieved by calcium phosphatemediated co-transfection of ccgM-cells with 5-10 μg of plasmid pXuBaxA(FIG. 1, section D) and 2 μg of DNA of HΔgM-lacZ or HΔgM-GFP,respectively.

Subsequent analysis of DNA of HΔgM-GFP with a digoxigenin labeled probespecific for the BamHI-HindIII fragment of plasmid pBH3.1 (FIG. 2)revealed

(i) a 2.757 bp and a 9.043 bp fragment on BamHI,

(ii) a 10.006 bp and a 825 bp fragment on HindIII,

(iii) and to a 5.415 bp and a 4.474 bp fragment on PstI digested DNA.

The restriction enzymes used (BamHI, HindIII, PstI) do cut withinsequences of the GFP-marker cassette, thereby altering the fragmentpattern relative to the respective GFP-marker cassette free DNA.

The GFP-probe bound to the respective first fragments (i-iii) and didnot detect GFP-specific sequences on DNA of RacH or of HΔgM-w.

On DNA of RacH the expected BamHI (11.166 bp), HindIII (10.199 bp) andPstI (9.257 bp) fragments were detected, which decreased in size afterremoval of 962 bp of gM-sequences accordingly (to: 10.204 bp, 9.237 bpand 8.279 bp; FIG. 3, section B).

Single-step growth kinetics of gM-negative viruses (HΔgM-w or HΔgM-GFP),which had been constructed as described in the legend to FIG. 1, andRacH were performed. Rk13 cells in 24 well plates were infected at anMOI of 2 of the respective viruses. Supernatants and infected cellpellets were harvested separately at various times p.i. (0, 4, 8, 12,16, 20, 24 h p.i.). Supernatants were cleared of cellular debris by lowspeed centrifugation and cells were subjected to freeze-thawing beforecell-associated infectivity was assayed. All virus titers weredetermined individually on Rk13 or ccgM cells, respectively, in 24 wellplates. The results (data not shown) can be summarized as follows:Cell-associated infectivity of both HΔgM-viruses was reduced betweenfactor 1.6 (4 h p.i.) and 45 (20 h p.i.) on Rk13 cells when compared totiters of cells infected with RacH (intracellular infectivity).Extracellular virus titers of both the HΔgM viruses were maximallyreduced by 186 (HΔgM-w) or 650 (HΔgM-GFP) fold (12 h p.i.) compared tothose of RacH (extracellular infectivity), supporting a role of gM invirus egress of RacH also.

Example 2 gM Deleted EHV-4 Isolates

To parallel the construction of gM-negative EHV-1, lacZ selection waschosen for selection of EHV-4. To allow the isolation of a gM-negativeEHV-4, a Vero cell line constitutively expressing EHV-4 gM wasconstructed. Vero cell clone C1008 (ATCC number: CRL-1586 was maintainedin minimal essential medium supplemented with 5-10% fetal calf serum(Biochrom, γ-irradiated). Recombinant cell line Vero-gM was generated byEffectene™ (Qiagen) mediated transfection of 1 μg of plasmid pCgM4 (FIG.3, section B) and 0.1 μg of plasmid pSV2neo (conferring resistance toG418; Neubauer et al., 1997) into Vero cells. Cell clones were firstselected for resistance to G418 (Calbiochem), then fortrans-complementation of a gM negative EHV-4. G418 was added to themedium of every 5th passage of recombinant cell lines (500 μg/ml). Allcells were regularly analyzed for Mycoplasma by PCR and for Bovine ViralDiarrhea Virus (BVDV) antigen by FACS analysis. The selected cell clonewas called Vero-gM and used in the following experiments.

EHV-4 DNA was cotransfected with plasmid pgM4β (FIG. 3, section B) intoVero-gM cells. The recombination resulted in several “lacZ-positive”plaques, that were isolated and replated. But then the purification of adeletion mutant in EHV-4 turned out to be more difficult and slower thanin EHV-1 as: (i) “lacZ-positive” plaques generally stained less intenseon Vero cells than on Rk13 cells and were thus harder to identify, andas (ii) the EHV-4 system replicated slower than EHV-1 and thus gave lesstime between plaque identification and isolation of viable virusprogeny.

All lacZ-positive plaques, that were isolated, were lost over the firstround of purification, which made it imperative to search for anothersolution. It was therefore attempted to use the GFP-marker for EHV-4also. In a first step, plasmid pgM4GFP+ (FIG. 3, section B) wasconstructed and used for recombination into EHV-4 DNA. ResultingGFP-positive plaques were purified by three rounds of isolating singleplaques on cell line Vero-gM. A homogenous GFP-positive virus populationwas chosen and viral DNA subjected to Southern blot analysis (FIG. 4).DNA of the resulting virus, E4 gM-GFP (FIG. 3, section C), was thencotransfected with either plasmid pgM4R or pgM4w (FIG. 3, section B).Plasmid pgM4R was used for the construction of an gM-repaired EHV-4,called E4RgM (FIG. 3, section A). Plasmid pgM4w was the basis togenerate the simultaneously gM- and marker gene-negative EHV-4, E4 gM-w(FIG. 3, section D). E4RgM and E4 gM-w virus populations were isolatedfor a GFP-negative phenotype, purified on Vero or on Vero-gM cells andfinally analyzed by Southern blot (FIG. 4). To express EHV-4 gM ineukaryotic cells plasmid pCgM4 was generated (FIG. 3, section B). Thecomplete ORF of EHV-4 gM was amplified by PCR using primers given inFIG. 8 and the Taq polymerase (MBI-Fermentas). The resulting PCR-productwas inserted into vector pcDNAI/Amp (Invitrogene).

Plasmid pgM4R resulted after PCR-amplification of nucleotides 91.699 to94.808 (Telford et al., 1998) (SEQ ID NO:2) of EHV-4 and insertion ofthe amplification product into vector pGEM3Zf+ (Promega) (FIG. 3,section B and FIG. 8). This plasmid was used for the construction of thegM-repaired EHV-4, E4RgM (FIG. 3, section A).

To construct plasmid pgM4β+ (FIG. 3, section B), that was designed toinitially delete 1110 bp of the 1352 bp EHV-4 gM, a multistep strategywas chosen. In a first step, both the flanking sequences necessary forDNA recombination were amplified independently by PCR using the PFUpolymerase (Stratagene). Restriction sites necessary for stepwisecloning were added by primer sequences (FIG. 8). PCR products wereinserted into vector pTZ18R (Pharmacia), resulting in plasmids pgM4Del1and pgM4Del2 (FIG. 8).

In a next step the 3.9 kbp E-coli lacZ expression cassette was releasedfrom plasmid ptt264A+ (Osterrieder et al., 1996) by SalI and BamHIdigest and was inserted into plasmid pgM4Del1 resulting in plasmidpgM4Del1β+. Then, EHV-4 specific sequences, taken out of pgM4Del2, wereintroduced into pgM4Del1+ using PstI and SalI, such that the marker genecassette was flanked by EHV-4 specific sequences in the resultingplasmid pgM4β+ (FIG. 3, section B).

Plasmid pgM4β+ was then digested with SalI and BamHI, again releasingthe lacZ-cassette, the resulting 5′-overhangs were filled in with theKlenow polymerase and the construct resulting from religation was calledpgM4w (FIG. 3, section B). Again a frame shift between the N-terminal208 nt and the remaining C-terminal 33 nt of the gM-sequence wasdesigned.

To generate plasmid pgM4GFP+ (FIG. 3, section B), the lacZ-cassette wastaken out of pgM4β+ (FIG. 3, section B) using the SalI and BamHI sitesand replaced by the GFP-cassette (including CMV-promoter andSV40-polyA), that had been removed of vector pEGFP-C1 (Clontech) viaAsnI and MluI digest. Restriction enzyme generated overhangs were bluntend adjusted by the Klenow polymerase.

Correct amplification of all PCR products was confirmed by cyclesequencing (MWG Biotech) and comparison to the published sequence ofEHV-4 strain NS80567 (Telford et al., 1998) (SEQ ID N0:2).

Recombination into EHV-4 was done by using the cell line Vero-gM andeither plasmid pgM4β+ or pgM4GFP+ (FIG. 3, section B) was cotransfectedwith EHV-4 DNA to generate a first generation gM-negative EHV-4 (FIG. 3,section C). Plasmid pgM4w (FIG. 3, section B) in combination with DNA ofthe gM-negative EHV-4 resulted in E4ΔgM-w (FIG. 3, section D). Finally,the gM-repaired EHV-4, E4RgM (FIG. 3, section A), was isolated afterco-transfection of plasmid pgM4R (FIG. 3, section B) with DNA ofE4ΔgM-GFP into Vero-cells.

DNA of EHV-4, E4RgM, E4ΔgM-w and E4ΔgM-GFP were cleaved with PstI, EcoRVor HindIII and DNA-fragments blotted onto nylon membranes. Parallelmembranes were either hybridized with GFP-specific sequences(identically to FIG. 2) or with a probe, named gM3.1, containing theEHV-4 specific sequences taken out of plasmid pgM4R (FIG. 3, section B).

Digestion results obtained (FIG. 4) were as follows:

-   -   (i) On DNA of E4ΔgM-GFP the GFP-probe recognized fragments at        5.531 bp, when PstI cleaved, at 8.383 bp after EcoRV-digest and        at 4.528 bp after HindIII digest. Identical fragments plus        fragments at 1.792 bp (PstI), 1.801 bp (EcoRV) and 826 plus        5.487 bp (HindIII) reacted with probe gM3.1.    -   (ii) Neither parental EHV-4, nor the repaired virus E4RgM or        E4ΔgM-w carried any GFP specific sequences.    -   (iii) The gM3.1 reactive DNA-fragments in EHV-4 and E4RgM were        detected at 6.806 bp (PstI), at 7.874 bp plus 1.801 bp (EcoRV)        and at 4.837 plus 5.487 bp (HindIII), respectively.    -   (iv) The gM- and GFP-cassette negative virus E4ΔgM-w, did not        hybridize with GFP-sequences, but with the respective EHV-4        specific sequences (gM3.1). The latter probe detected fragments,        lacking 1110 bp of gM sequences, when compared to wild type        virus, i.e. at 5.696 bp (PstI), at 6.764 bp plus 1.801 bp        (EcoRV) and at 3.727 bp plus 5.487 bp (HindIII), respectively.

On HindIII cleaved DNA of all of these viruses another gM3.1 probespecific fragment exists at 126 bp (FIG. 2), but this fragment is tosmall to be shown in this Southern blot.

Viruses E4ΔgM-GFP and E4ΔgM-w lost their capacity to express gM and inaddition E4ΔgM-w lost the marker gene sequence.

-   -   a) Virus growth on culture cells. Virus growth properties of the        various mutant viruses as detailed above were compared on Vero        and on Vero-gM cells. Cells seeded in 24 well plates were        infected at an MOI of 1-2 and extracellular (extracellular        infectivity) and intracellular (intracellular infectivity) virus        titers were determined at different time points p.i. (FIG. 5).        While growth properties of the rescuant E4RgM virus corresponded        well to those of EHV-4, there was a surprising inhibition in the        production of E4ΔgM-w and E4ΔgM-GFP extracellular virus titers        on non-complementing cells. Within this series of experiments        (mean of two independent experiments) extracellular infectivity        could never be detected before 24 h p.i. Even at 30 hours p.i.        only very low titers were extracellularly observed (maximum of        1.5 plaques/ml at the lowest dilution 10-1), although cells        showed severe cytopathic effect. Differences in intracellular        infectivity, however, never reached 100 fold and peaked at 24        hours p.i. (84 fold between EHV-4 and E4ΔgM-w). The delay in        detecting intracellular infectivity was only one time point (12        h versus 15 h. p.i.). Taken together it could be surprisingly        demonstrated that deletion of gM-sequences of the EHV-4        background massively influenced virus replication in vitro, but        that expression of gM is not essential for replication.        Especially extracellular infectious virus decreased and the        ability to directly infect adjacent cells was diminished—as        reflected by plaque sizes.    -   b) Plaque size. Diameters of 150 plaques after infection of Vero        or Vero-gM cells with EHV-4, E4RgM, E4ΔgM-w or E4ΔgM-GFP were        measured and average plaque sizes were determined relative to        sizes of wildtype plaques, that were set 100%. It was clearly        demonstrated, that the gM negative viruses were able to infect        and replicate in Vero cells, but that the maximal plaque        diameters were markedly reduced, to less than 20% of wildtype        plaque diameters (FIG. 6). Infection with the parental or the        rescuant virus resulted in wild-type-like appearance of plaques,        indicating that the observed phenotype was indeed induced by the        gM-deletion. This was additionally corroborated by the fact,        that plaque formation of E4ΔgM-w and E4ΔgM-GFP was fully        restored on cell line Vero-gM (Data not shown).    -   c) Penetration experiments. In this experiment, the influence of        the EHV-4 gM on entry kinetics of EHV-4 was assessed. 100 PFU of        the different viruses, parental EHV-4, the gM repaired virus        E4RgM, as well as the gM-deletion mutants and E4ΔgM-GFP (see        FIG. 3), were allowed to adsorb at 4° C. to Vero cells in 6-well        plates. After 90 min, the respective inocula were replaced by        fresh medium and penetration was initiated by shifting the        incubation temperature to 37° C. At different times after the        temperature shift—starting immediately (=0 min)—extracellular        infectivity was pH-inactivated by treating cells with a citrate        buffer (pH 3.0). Parallel samples were washed accordingly, but        the citrate buffer was replaced by PBS, such that at every time        point the “adsorbed infectivity” could be compared to the        “penetrated infectivity”. Numbers of plaques were determined        after incubating cells for four days under a methocell overlay.    -   d) Several sets of experiments were performed: In FIG. 7A        results are depicted for genotypically and phenotypically        gM-negative viruses after propagation on non complementing Vero        cells, whereas FIG. 7B represents kinetics of phenotypically        complemented E4ΔgM-w and E4ΔgM-GFP, as viruses had been grown on        gM-expressing Vero-gM cells.

A mean of 52.8% (56.7%) of the infectious parental EHV-4 (E4RgM) wasprotected from extracellular acid treatment at 40 min after initiatingpenetration (FIG. 7A, open circles) whereas only 33.7% (E4ΔgM-w—closedrectangles) and 38.5% (E4ΔgM-GFP—closed triangles) of gM negativeviruses were protected, yet. At later time points of entry kinetics, thedifferent graphs start to variously overlap and a maximal penetrationefficiency between 61.7% and 78.9% is reached after 150 min ofpenetration time, indicating that a certain assay variability mayaccount for the slight differences observed.

When gM negative viruses had been prepared on complementing Vero-gMcells no difference at all was observed in their penetration efficiency(FIG. 7). As opposed to a delay in entry kinetics of gM-negative EHV-1of about 20% (strain RacL11; Osterrieder et al., 1996) to up to 40%(strain KyA; Seyboldt et al., 2000), the effect of deleting gM of EHV-4has to be noted with reservation. Nevertheless the following conclusionscan be drawn: (i) A difference was observed in kinetics ofphenotypically complemented to non complemented gM-negative viruses(FIGS. 7A and 7B), but (ii) the influence of deleting gM on EHV-4penetration is virtually undetectable.

Example 3 Analysis of Horse Sera for Anti gM Antibodies Using a gMSpecific Serological Test

To state whether gM can be used as a serological marker for distinctionof wild type infection versus vaccination with a gM-negative vaccine,several assumptions had to be tested. Primarily it needed to be assessedwhether sera of field virus infected horses contain gM-specificantibodies. For initial analysis a Western blot test was chosen, as thissystem allowed to identify a specific reaction against backgroundreactivity. When using highly neutralizing sera (EHV-1 and/or -4neutralizing titers between 1:128 and 256), it was established (data notshown) that lysates of EHV-1 gM expressing cell line ccgM allowed thedetection of a specific signal by horse sera and that a dilution of serato 1:3000 seemed to work best.

Consequently, sera of all 12 foals (6 vaccinates and 6 controls) thathad participated in an EHV-1 gM vaccine trial were analyzed for gMreactivity in Western blot. Of each individual horse three differentsera were tested: Taken before entering the trial (Pre), 4 weeks afterthe second vaccination (V2) and two weeks after challenge infection (C),respectively.

In summary by Western blot analyses (FIG. 9) it was shown that (i) seraof horses, exhibiting EHV-1 neutralization activity, all tested positivefor gM, that (ii) gM reactivity was never detected in any of the samplesanalyzed before known contact to EHV-1 or after vaccination with thegM-negative EHV-1 and that (iii) after infection of vaccine trial horseswith the gM-positive challenge virus, gM was clearly detectable in 10out of 12 cases. Finally (iv) it was observed that anti EHV-1 antibodytiters and the intensity of gM reactivity seemed not to be directlycorrelated.

Due to the high background reactivity of horse sera, the establishmentof serological tests is difficult. Based on indirect immunofluorescence(IIF) data obtained in horse sera, it was confirmed that either anindirect or a blocking test system will have to be established or highlypurified gM-polypeptides need to be used in an ELISA test. To this end,an ELISA was established as follows. Either purified gM polypeptides orthe complete gM was immobilized onto the solid support of a 96 wellplate which was coated to ensure good attachment of the capturingprotein. For the assay, unspecific binding sites were be blocked byeither dry milk or similar substances to prevent unspecific binding.Following this, the plastic surface was rinsed with an appropriatewashing buffer (e.g., PBS-Tween) to remove excess blocking agent. Thenthe test samples were added to selected wells and the ELISA plate wasincubated at 37° C. according to standardized methods, allowingantibodies in the test sample to bind to the immobilized capturingprotein. In the next step, the wells of the ELISA plate were washedthoroughly by several times rinsing with washing buffer, followed by theaddition of a suitable anti-horse antibody coupled to an enzyme such asHRP (horse radish peroxidase). The detection of bound antibody/HRPconjugate was finally performed by adding a suitable substrate, followedby photometric read-out of optical density of individual wells. Thevalue obtained was be compared to positive and negative controls run inthe same assay.

Example 4 Identification of EHV-4 gM

Although the predicted amino acid sequence of EHV-4 gM is calculated tobe 86.7% identical to that of EHV-1 gM (Telford et al., 1998), antiEHV-1 gM Mab 13B2 (Allen and Yeargan, 1987) specifically reacts inWestern blot with the type-specific protein only (Crabb et al., 1991).To nevertheless identify the EHV-4 homolog in this study, otheranti-EHV-1 gM antibodies (Seyboldt et al., 2000; Day, 1999) were testedon purified EHV-4 virions, on lysates of cells infected with EHV-4 or onlysates of Vero-gM cells. The latter being a recombinant cell linedeveloped to synthesize EHV-4 gM under control of the IE-HCMV promoter.The reactivity of all anti-EHV-1 gM monoclonal antibodies against EHV-4gM was below the detection limit in Western blot, whereas parallel EHV-1samples were always readily reactive (data not shown). Only thepolyclonal antiserum, that had been generated in rabbits against aHis-tagged EHV-1 gM derived polypeptide (amino acid 376-450; Seyboldt etal., 2000), reacted strong enough with the heterologous gM to allow theidentification of EHV-4 gM (FIG. 10, section A). Using this antibody aspecific reactivity at an Mr of about 50,000 to 55,000 was observed inpurified EHV-4 virions. According to its predicted hydrophobicproperties the detected gM-protein aggregated upon boiling. In contrastthe form of gM expressed in recombinant Vero-gM cells mainly run at anMr of about 46,000 to 48,000, indicating that the gM-proteins of EHV-4are processed similarly as has been shown for EHV-1 (Osterrieder et al.,1997; Rudolph and Osterrieder, 2002).

Several experiments were conducted to analyze the phenotype of thegM-deletion in EHV-4. To compare expression of other glycoproteins,lysates of Vero cells infected with EHV-4, E4RgM, E4ΔgM-w or E4ΔgM-GFPwere subjected to Western blot analysis. It is demonstrated that thedeletion of gM did not influence the production of the late proteins gBor gD, indicating that early steps in virus replication were notsubstantially affected by the deletion.

In another experiment it could be demonstrated by analysis of virionpreparations of wildtype, repaired or both gM-deleted EHV-4, that no gMreactivity at all was detectable within gM-negative viruses, whereas theprotein was readily reactive in control virions. The presence of virionsin the respective preparation was shown in a parallel blot probingagainst gB (FIG. 10, section B).

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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What is claimed is:
 1. A recombinant Equine Herpes Virus type 4(EHV-4),wherein 900-1150 bp of the gM open reading frame are deleted, whereinsaid EHV-4 is free of heterologous elements.
 2. A recombinant EquineHerpes Virus type 4 (EHV-4) wherein the entire gM coding sequence isdeleted except for 0-50 bp of the coding sequence for the C-terminalportion and except for 150-250 bp of the coding sequence for theN-terminal portion, and wherein said EHV-4 s free of heterologouselements.
 3. The EHV-4 according to claim 2, wherein the entire gMcoding sequence is deleted except for 34 bp of the coding sequence forthe C-terminal portion and except for 209 bp of the coding sequence forthe N-terminal portion.
 4. A recombinant Equine Herpes Virus type 4(EHV-4) wherein nucleotides 92681-92731 to 93765-93865 of the gM codingsequence as corresponding to SEQ ID NO:2 are deleted, and wherein saidEHV-4 is free of heterologous elements.
 5. The EHV-4 according to claim4, wherein nucleotides 92681 to 93865 of the gM coding sequence ascorresponding to SEQ ID NO:2 are deleted.
 6. The EHV-4 according toclaim 4, wherein nucleotides 92715 to 93824 of the gM coding sequence ascorresponding to SEQ ID NO:2 are deleted.
 7. The EHV-4 according toclaim 1, wherein said EHV-4 is based on MSV Lot 071398 and isolateE4ΔgM-w and that it is the EHV-4 which was deposited at the ECACC/CAMRon Jan. 14, 2003 with the accession number 03011401.